The tools of molecular biology have enabled researchers to introduce segments of DNA from one organism into another organism. As the need for the introduction of multiple segments of DNA and larger fragments of DNA into numerous target hosts increases, the need for novel cloning strategies and improved vector constructs increases accordingly.
Vectors for Agrobacterium-mediated transformation of plant cells typically comprise restriction cloning sites, T-DNA border elements from the tumor-inducing plasmid of Agrobacterium tumefaciens, a gene of interest, some type of selectable marker, and at least one bacterial origin of replication. These vectors are then introduced into plant cells to transform them with the gene of interest.
There is a need in the art for vectors that are relatively small for convenience of manipulation and capable of prolific replication in a living cell enabling the amplification of the inserted donor DNA. There is also need in the art for vectors with convenient restriction sites that can be used for insertion of the DNA to be cloned. It is also desirable that there be a mechanism for easy identification, recovery and sequencing of the recombinant molecule.
It is common to develop plant transformation vectors from earlier-used vectors with the result that vector constructs typically contain unwanted or unneeded restriction sites and unnecessarily large stretches of polynucleotide that serve no purpose other than to link essential modular elements of a construct.
As plant transformation becomes more widely used, especially in high throughput screening of transgenic constructs, there is an increased need for technology to track both specific vector constructs and general transformation events during all stages of development, i.e. from the cloning of a single bacterial cell with a gene of agronomic interest, through the testing of a seed of a fertile transgenic plant that may eventually contain the vector construct for both quality control and regulatory purposes.